Apparatus for Pathogen Detection

ABSTRACT

An apparatus for separating an analyte from a test sample, such as bacteria from blood components, based on their dielectric properties, localizing or condensing the analyte, flushing substantially all remaining waste products from the test sample, and detecting low concentrations of the analyte. The module array includes a plurality of microfluidic channels with connecting microfluidic waste channels for directing undesired material away from the analyte. An electric field is applied causing a positive dielectrophoretic force to the analyte to capture the analyte. The electric field is applied to at least one electrode having a plurality of concentric rings or concentric arcs extending radially outwards from a center point, electrically connected to a voltage source such that when voltage is applied to the at least one electrode, the concentric rings or concentric arcs alternate in voltage potential.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to an apparatus for pathogen detection.Specifically, the invention relates to the field of pathogen detectionsystems and diagnostic devices and their micro-component assembly. Morespecifically, the invention utilizes an apparatus that includes adielectrophoretic separator, a dielectrophoretic condenser, adielectrophoretic trap, microfluidic components, and field effectsensor, such as an ion sensitive sensor, nanowire sensor, or nanoribbonsensor configured as biosensors, to perform a pathogen detectionprocess. The invention further relates to a specific electrode designfor high yield pathogen and cell capture and separation.

2. Description of Related Art

Bacterial infections cause thousands of diseases in humans and animalsevery year. Recent deadly outbreaks of E. coli, Salmonella, and Listeriahave highlighted the urgent need for more effective methods ofdetection, identification, and characterization of pathogens, and theirorigin and proliferation. Conventional detection methods have proveninadequate because they suffer from long incubation periods, high cost,and require highly trained personnel to operate. There remains a strongneed for a reliable, time-efficient apparatus and method for specificdetection of bacteria in low concentration.

Conventional methods rely on bacterial culture growth, which requirehighly qualified personnel and time, both contributing to higher costsfor the procedure. The most widely used method for bacterial detection,the standard plate count, takes from 24 to 48 hours due to the timeneeded for bacteria to grow detectable colonies, and requires a stockedmicrobiology lab. Although faster methods, such as PCR (Polymerase ChainReaction) Plates or labeled detection and fluorescent imaging, canreduce the response time to one hour, these require complex samplepreparation, highly trained personnel, high cost per test, and havelimited portability.

The major challenge in automated sample preparation for detection fromblood or other unprocessed liquids using microstructures is efficientseparation of the analyte of interest (bacteria, cells, or particles)from large blood components. Red blood cells (RBC) and white blood cells(WBC) range between 6 μm-21 μm in size and constitute over 50% of thewhole blood volume. RBC and WBC presence obstructs the detection ofbacteria, cells, or particles. The present invention is a miniaturizeddevice for rapid pathogen screening that overcomes these obstacles.

Dielectrophoresis (“DEP”) is a separation method based on size anddielectric properties and has been described in literature as forexample in Pohl et al, Science 1966, and Sher Nature 1968, Voldman,Annual Review Of Biomedical Engineering, 2006. The use of DEP tomanipulate particles and cells has been previously described, as forexample, in H. Pohl, I. Hawk, “Separation Of Living And Dead Cells ByDielectrophoresis,” Science, 152, 3722 (1966); Y. Huang, R. Holzel, R.Pethig, X. Wang, “Differences In The Ac Electrodynamics Of Viable AndNon-Viable Yeast Cells Determined Through Combined Dielectrophoresis AndElectrorotation Studies,” Phys. Med. Bid., 37, 7 (1992); S. Chang, Y.Cho, “A Continuous Size-Dependent Particle Separator Using A NegativeDielectrophoretic Virtual Pillar Array,” Lab Chip, 8, 1930-1936 (2008);and J. Yang, Y. Huang, X. Wang, F. Becker, P. Gascoyne, “DifferentialAnalysis Of Human Leukocytes By DielectrophoreticField-Flow-Fractionation,” Biophysical Journal, 78, 2680-2689 (2000).However, effective methods for cell/pathogen separation on a micro-scalefrom fluids containing pollutants of comparable size are stillunattainable.

High-frequency electric fields when applied to an electrically neutralobject cause polarization. A high-frequency non-uniform electric fieldgives rise to a dielectrophoretic force (DEP) F_(DEP) which acts on theobject.

A spherical object of a given electrical permittivity ε_(p) placed in amedium of a different permittivity ε_(m) in a spatially varying electricfield E(x,ω) is subjected to a dielectrophoretic force, F_(DEP). Thedielectrophoretic force is given by:

F _(DEP)=2πε_(m) r ³ Re{CM(ω)}·∇E ²

where

CM(ω) is the Clausius-Mossotti factor=(ε_(p) ^(˜)-ε_(m) ^(˜))/(ε_(p)^(˜)+2ε_(m) ^(˜))

ε^(˜)=ε+σ/iω;

Re{CM(ω)} is the real part of the CM(ω), which can be a complex number;

ε_(p) is the particle permittivity;

ε_(m) is the permittivity of the liquid medium;

r is the particle radius;

ε^(˜)is the complex permittivity (complex dielectric function);

σ is the conductivity;

ω is the angular frequency; and

∇E is the gradient of the electric field.

Depending on the respective permeability (ε^(˜)) and conductivity (σ) ofthe object and the medium, the force can be attractive (positivedielectrophoresis (pDEP)), or repulsive (negative dielectrophoresis(nDEP)). If Re{CM(ω)} is positive, then the particle experiences apositive dielectrophoretic force, and if Re{CM(ω)} is negative, then theparticle experiences a negative dielectrophoretic force. Differentspecies have different dielectric properties. The dielectric functionsε_(m), ε_(p) depend on the frequency of the external electric field. Thepermittivity of the medium affects the CM(ω) factor and the value ofRe{CM(ω)}. Importantly, if the signs of Re{CM(ω)} for different speciesare opposite then the species are subject to forces acting in oppositedirections and separation occurs.

There is a cross-over frequency, ω_(co), that occurs when the Re{CM(ω)}goes to zero. Critical to separation is that ω_(co) is uniquelydifferent for different cells and bacteria. Separation procedures forstained cells have been described in U.S. Pat. No 7,153,648 entitled“Dielectrophoretic Separation Of Stained Cells,” where appropriatefrequency and amplitude are applied via a function generator, and redblood cells are attracted to electrodes by positive dielectrophoresisforce, while stained white blood cells are repelled to the area withweakest electric field by negative dielectrophoresis force. Thedifferential behavior and separation of E. coli cells from human bloodcells on electrodes under applied electric field has been described inpatent U.S. Pat. No. 6,989,086 entitled “Channel-less separation ofbioparticles on an Electronic Chip by Dielectrophoresis.”

The dielectrophoretic force is affected both by the geometry of theelectrodes (gradient of the electric field), the Re{CM(ω)} factor, anddepends on the dielectric constant of the medium ε_(m).

Using the aforementioned prior art techniques for dielectrophoresis, theseparation of bacteria from blood may achieve, at best, an efficiency ofapproximately 30%.

SUMMARY OF THE INVENTION

Bearing in mind the problems and deficiencies of the prior art, it istherefore an object of the present invention to provide a filtrationsystem for pathogen detection that utilizes a plurality ofdielectrophoretic modules with distinctive functionality and geometry toobtain separation performance which cannot be obtained using the singlemodule apparatus of the prior art.

It is another object of the present invention to provide a pathogendetection system that includes a capture/release mechanism for solutionexchange without cell loss to enhance pathogen detection at lowconcentrations.

It is yet another object of the present invention to provide a methodfor separating a low concentration of bacteria (or otherpathogens/particles) from a high volume of blood (or other fluids) whichis based on the dielectric properties of the products. The speciesseparation being enhanced and promoted by dielectrophoretic forcesacting on the test sample in a plurality of microfluidic channels.

It is another aspect of the present invention to provide a filtrationsystem for pathogen detection that can accommodate high and lowthroughput, capable of processing test sample volumes significantlygreater than micro- or pico-liters, yet capable of processing the minutetest sample volumes as well.

It is yet another object of the present invention to provide anelectrode design that can perform high yield pathogen and cell capture,and enhance separation. It is another object of the present invention toprovide pathogen detection in liquid media, especially for use with foodand agricultural products to improve health standards at the consumerlevel.

It is another object of the present invention to provide time sensitivepathogen detection for point-of-care diagnostics.

Still other objects and advantages of the invention will in part beobvious and will in part be apparent from the specification.

The above and other objects, which will be apparent to those skilled inthe art, are achieved in the present invention which is directed to anapparatus for pathogen detection comprising: a first chamber for storinga test sample including product to be analyzed and microscaledcomponents to be separated from said product to be analyzed; a secondchamber for storing a reference solution; a pump for pumping said testsample and said reference solution; a microfluidic separator separatingsaid product to be analyzed from said microscaled components, saidmicrofluidic separator including a plurality of microfluidic channels,each microfluidic channel including: at least one electrode forproducing a dielectrophoretic force on said test sample when said testsample is pumped through said microfluidic channel to perform adielectrophoresis-based separation, said at least one electrodecomprising: a plurality of concentric rings or concentric arcs extendingradially outwards from a center point, electrically connected to avoltage source such that when voltage is applied to said at least oneelectrode, said concentric rings or concentric arcs alternate in voltagepotential, wherein each odd numbered ring or arc counted from saidcenter point is held to a first voltage potential, and each evennumbered ring or arc is held to a second voltage potential, said firstvoltage potential different from said second voltage potential inmagnitude, phase, polarity, or any combination thereof; and channels fortransporting said microscaled components away from said product to beanalyzed.

The apparatus further includes a third chamber for storing saidmicroscaled components when separated from said product to be analyzedby said plurality of microfluidic channels; a condenser for capturingsaid product to be analyzed once said product has passed through saidmicrofluidic channels and is substantially separated from saidmicroscaled components; and a sensor for detecting said product to beanalyzed.

The plurality of microfluidic channels may be assembled in an array,each microfluidic channel having at least one electrode on an internalwall for delivering the dielectrophoretic force to the test sampletraversing through the microfluidic channel.

The microfluidic channels preferably comprise a plurality of plates,such that each microfluidic channel represents an elongated pathway forthe test sample capable of providing a dielectrophoretic force or forcearising from AC electric field in fluids to the test sample as the testsample traverses the microfluidic channel.

A collecting electrode is used to attract the product to be analyzed atan inlet of the sensor. The sensor includes a field effect based sensor,nanowire sensor, nanoribbon sensor, or ion sensitive field effecttransistor, and is capable of applying a confining dielectrophoreticforce, trapping the product to be analyzed.

The pump may be a micro-pump operating in tandem with micro-valves toachieve a fully automated pathogen detection filtration system capableof miniaturization to a chip-scale design.

The apparatus may also include a microfluidic transport module fortransporting the product to be analyzed to a location in the vicinity ofthe sensor.

The electrode design may include even numbered concentric rings or arcsin electrical communication with one another, and odd numberedconcentric rings or arcs are in electrical communication with oneanother.

In a second aspect, the present invention is directed to an apparatusfor pathogen detection comprising: a microfluidic assembly including aplurality of microfluidic channels forming an array, each of saidmicrofluidic channels including: at least one electrode for establishingdielectrophoretic forces on a test sample separating portions of saidtest sample into an analyte and a waste product, said at least oneelectrode comprising a plurality of concentric rings or concentric arcsextending radially outwards from a center point, electrically connectedto a voltage source such that when voltage is applied to said at leastone electrode, said concentric rings or concentric arcs alternate involtage potential, wherein each odd numbered ring or arc counted fromsaid center point is held to a first voltage potential, and each evennumbered ring or arc is held to a second voltage potential, said firstvoltage potential different from said second voltage potential inmagnitude, phase, polarity, or any combination thereof; adjacentmicrochannels for receiving said waste product attracted by adielectrophoretic force, removing said waste product from said analyte;a condenser including an electrode for localizing said analyte forsensing; and a sensor for detecting said analyte.

A pharmaceutical or other substance may be introduced which piercesmembranes of an alive analyte component in the reference solution at apredetermined frequency, but not membranes of other analyte componentsor dead analyte components, and differentiating the alive component fromother analyte components and dead analyte components throughdielectrophoretic forces.

The pH or conductivity of the test sample may be adjusted for control ofvoltage and frequency dependence for the Clausius-Mossotti factorcross-over frequency.

Change of the Clausius-Mossotti factor cross-over frequency may beinduced for the analyte by adding or mixing an additional fluid.

The step of detecting low amounts of analyte using a microfluidic sensorincludes using an electric field at predetermined flow conditions, toimmobilize the analyte on the surface of a field effect based sensor,nanowire sensor, nanoribbon sensor, or ion sensitive field effecttransistor sensor.

Super positioning or tuning of frequency components, waveform shapes,and waveform tunings, or any combination thereof, may be performed tomaximize separation, differentiation, capture, or release of theanalyte.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the invention believed to be novel and the elementscharacteristic of the invention are set forth with particularity in theappended claims. The figures are for illustration purposes only and arenot drawn to scale. The invention itself, however, both as toorganization and method of operation, may best be understood byreference to the detailed description which follows taken in conjunctionwith the accompanying drawings in which:

FIG. 1 depicts a filtration system 10 of the present invention forpathogen detection;

FIG. 2 is a schematic view of a microchannel assembly having a pluralityof individual microchannels;

FIG. 3 is a schematic view of an array of microfluidic microchannelassemblies of the present invention used for separation;

FIG. 4 depicts a cross-section of microchannel with ground electrodesand waste collecting electrode;

FIG. 5 depicts a computer generated model of the resultingdielectrophoretic forces F_(DEP) acting on bacteria in microfluidicchannels;

FIG. 6 depicts a computer generated model of the resultingdielectrophoretic forces acting on red blood cells in microfluidicchannels;

FIG. 7 is a cross-sectional top view of a microchannel showing thetrajectories of the test sample components.

FIG. 8 depicts a table (Table I) providing values of the coefficient αfor RBC, WBC, and bacteria at 10 MHz in blood serum;

FIG. 9 depicts a computer generated model of bacteria and red blood celltrajectories upon applied dielectrophoretic force in a microfluidicchannel;

FIG. 10 depicts a table (Table II) providing values for the real andimaginary permeability as well as the particle radius and conductivityof E. coli and Micrococcus in a reference solution and blood serum;

FIG. 11A depicts a graph of a simulation of the dielectrophoretic forceon E. coli inside a microfluidic channel as a function of channelposition and travel time;

FIG. 11B depicts a graph of a simulation of the dielectrophoretic forceon RBCs inside a microfluidic channel as a function of channel positionand travel time;

FIG. 12 depicts a set of electrodes and their respective geometry in across-section of a microfluidic channel for capturing and immobilizingbacteria;

FIG. 13 depicts a force diagram of the resultant electrodes of FIG. 12showing the direction of the dielectrophoretic force acting on bacteria;

FIG. 14 depicts a flow velocity profile with inflow from left and thedielectrophoresis force directing/pushing bacteria to the sensor at thebottom of a microfluidic channel (overcoming diffusion limitations);

FIG. 15A depicts fabrication levels or steps of integration of thepresent invention on an integrated circuit chip;

FIG. 15B depicts an expanded assembly drawing of the layers representingthe fabrication steps of FIG. 15A;

FIG. 16 is an expanded assembly drawing of the layers of a microfluidicseparator;

FIG. 17 depicts electrical connections and microfluidic connectionsbetween components provided in embedded layers of an integrated circuitdevice of the present invention;

FIG. 18 depicts an embodiment of the electrode design 200 for use withinthe confines of a microchannel;

FIG. 19 depicts an electrode design having a set of adjacent concentricrings;

FIG. 20 depicts the electrode of FIG. 19 delineating alternatingpolarities associated with a positive voltage source connected to oneset of electrode rings, and a negative voltage source connected toanother set of electrode rings, where each ring of each set alternatesradially outwards from the center point;

FIG. 21 depicts a typical pathogen contaminated fluid inside amicrochannel of the present invention in the absence of an electricfield to the electrode;

FIG. 22 depicts the microchannel containing contaminated fluid of FIG.22 with the electrode's electric field ON;

FIG. 23 depicts the alignment of pathogens when a positivedielectrophoresis force (pDEP) of 3 volts at 10 MHz is applied to theelectrode; and

FIG. 24 depicts the absence of pathogen alignment when a negativedielectrophoresis force (nDEP) of 3 volts at 200 MHz is applied to theelectrode.

DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

In describing the preferred embodiment of the present invention,reference will be made herein to FIGS. 1-24 of the drawings in whichlike numerals refer to like features of the invention.

The filtration system of the present invention performs pathogendetection using a plurality of dielectrophoretic modules of microfluidicchannels with distinctive functionality and geometry to obtainseparation performance which cannot be obtained in the prior art.Additionally, the present invention integrates a nano-scaled sensor withthe filtration system. Advantageously, all components of the filtrationsystem may be embedded, forming an integrated electronic-microfluidiccircuit.

The assembled filtration system automatically transports, separates,condenses, and detects low amounts of particles, cells, and bacteria, orthe like, from liquids in a portable configuration that minimizes falsepositives and negatives.

The present invention defines a robust method for separating bacteriafrom blood components based on their dielectric properties, localizingthe bacteria, flushing substantially all remaining by-products from thebacteria, which generally are on the order of micro-sized or microscaledcomponents, and detecting low concentrations of the bacteria. Theseparation is fast and reliable as species movement is caused by amodule array imparting opposing forces. The module array includes aplurality of microfluidic channels with connecting microfluidic wastechannels for directing undesired material away from bacteria. Theprocess enables separation of low concentration of bacteria or otherpathogens or particles from blood or other fluids, which then enablesdetection of the low concentration of these species. This is of greatimportance for medical diagnostics and determining food safety.

In a preferred embodiment, the invention includes an electronic devicecapable of detecting of a low number of bacteria or other pathogens orparticles from milliliter or larger volumes of different liquid media ona minute time scale by integrating a plurality of modules ofmicrofluidic channels capable of performing a dielectrophoresis-basedseparation, and incorporating a unique capture system usingfield-effect-transistor based biosensors.

Bacteria present in a sample even of different types will be subject toa dielectrophoretic force in one direction, whereas all large bloodcomponents red blood cells and white blood cells will be subject to adielectrophoretic force in another direction. Effective separationimproves the detection of pathogens. Without separation, bloodcomponents which constitute a vast majority of micro-sized particles inblood, would clog active sensor sites and prevent detection of lowconcentrations of bacteria present in the same sample.

Furthermore, many types of sensors cannot operate in high ionicsolutions such as blood plasma. Consequently, the solution containingthe sample to be sensed, most likely bacteria, has to be changed to amore suitable reference solution, such as a buffer. Often apre-concentration step is required. The capture/release mechanismpresented by the present invention provides an excellent method forsolution exchange without cell loss.

Electric field cell capture, release, and separation based on forcesacting in opposite directions allow precise control of cell separationwithout risk of cell loss or contamination. Advantageously, the presentinvention may be applied for separating any species of comparable sizein any liquid medium; however, bacteria separation from bloodcomponents, white blood cells and red blood cells, is illustrated forexemplary purposes, and represents a predominate utilization of thepresent invention.

FIG. 1 depicts a filtration system 10 of the present invention forpathogen detection. The system includes two injection chambers: a firstinjection chamber 12 containing a sample to be tested, such as blood,and a second injection chamber 14 containing a reference solution,buffer, or other liquid for flushing at a later point in the process,generally referred to as the by-product or waste-product. Injectionchambers 12, 14 are connected to micro-pump inlets which connect to amicrofluidic separator 16. Fluids will be pumped into microfluidicseparator 16 in a controlled manner by pump 18 such that the test sample(e.g., blood) will be pumped in first. As will be discussed in furtherdetail below, the microfluidic separator includes a plurality ofmicrofluidic channels assembled in an array fashion that usesdielectrophoretic forces to separate continually during transportcomponents of the test sample from one another. In the illustrativeexample, bacteria may be separated from red and white blood cells. Theseparated analyte (bacteria) is then condensed by a localized electricfield, and reference solution, such as a buffer fluid, is used toreplace and dilute or change the chemical composition of the blood serumthat reaches condenser 20.

Unlike the prior art, the microfluidic separator 16 of the presentinvention includes a plurality of channels that apply dielectrophoreticforces that are exerted on the particles, cells, bacteria, and/ormicro-scale components as they flow through the channels. Thedielectrophoretic field is carefully chosen such that the components ofinterest that flow through will experience an opposite force as comparedto the rest of the components or waste-products that are desired to beseparated out. The waste-products are drained throughout the processfrom the plurality of microfluidic channels to waste chamber 22 throughmultiple microfluidic channel outlets.

In this manner, the filtration system of the present invention iscomposed of modules/segments each tuned such that the class of objectsunder study (i.e., the analyte) has the same response. For example, allbacterial have an nDEP, which is in the middle of the response spectrum.An additional “filter” is then applied for increased accuracy oftargeting the analyte. The assembly of independent modules for thisapplication is comprised of multiple, but not necessarily continuous,wires. Microchannel outlets, waste channels, break the continuous wireconfiguration. Using this geometry, surface electrode configurations maybe employed, moving away from continuous conducting wire of the priorart.

In this example, isolated bacteria flows into a condenser chamber 24,which has a collecting electrode to attract the bacteria to the inlet ofa microfluidic sensor 26 containing sensor arrays. Movement of bacteriato the field-effect-transistor based sensor is enhanced using theelectric field and the dielectrophoretic force to overcome the diffusionlimitation of the motion. Furthermore, the present invention is capableof tuning the electric field such that only the particle of interestgets through, the remaining product is eliminated. Thus, detection islabel-free; it does not require sensor functionalization with specificantibodies.

The method is based on dielectrophoretic separation followed bydielectrophoretic concentration, and replacement or partial replacementor dilution of the original liquid with a reference solution. The nextstep is dielectrophoretic manipulation of bacteria to the sensor surfaceto overcome the diffusion limitation and enable bacteria contact withthe sensor surface for detection.

The device operation and automated sample preparation is described insome detail below. First, a test sample injection is distributed intothe system. This is performed by a pump that causes the automateddistribution of the test sample, placing the test sample in a pluralitymicrofluidic channels (microchannels) via capillary forces (porousmedia) and pump-pressure driven flow. Each of the plurality ofmicrofluidic channels are lined with electrode geometry capable ofestablishing an electric field and a dielectrophoretic force on the testsample. Separation within the microfluidic channels is then performed bythe dielectrophoretic force. In order to achieve adequate and efficientseparation, waveform tuning of the electric field is selected with theintention that two types of species are subjected to forces acting inopposite directions. The separation occurs within microfluidic separator16. The unwanted micro-scaled components and blood cells (waste-product)separated from the analyte (bacteria/cells/particles) are collected inwaste chamber 22. The separated analyte is then collected on condenser20. An electrode immobilizes the desired analyte material.

In order to remove the waste-product, extraneous serum and otherunwanted blood products, the remaining analyte is exposed to a referencesolution while held by the condenser electrode. In this manner theunwanted blood products are flushed away, replaced or at least partiallyreplaced, and/or diluted by the reference solution. The remaininganalyte is localized to a sensor surface. Dielectrophoretic manipulationof bacteria is used at the sensor surface to overcome the diffusionlimitation and enable bacteria contact with the sensor surface fordetection.

Preferably the sensor is of nanowire or nanoribbon technology, whichenables the filtration system of the present invention to be integratedon a semiconductor chipset. Once the final analyte is interrogated, theoutput may be digitized for automated data processing and readout.

In a preferred embodiment, a multi-step approach to filtration forpathogen detection is achieved using a plurality of dielectrophoreticmodules including a plurality of microfluidic channels in an arrayfashion. The microfluidic separator 16 separates the test samplecomponents of interest (e.g., bacteria) from pollutants (e.g., bloodcells and blood serum). In a subsequent process step, the surroundingmedium is then exchanged or diluted with a reference solution moresuitable for comprehensive electronic detection applications.

The process introduces condensation of the analyte onto a concentratingelectrode using a dielectrophoretic (pDEP) force. Once all remaininganalyte from the sample is collected on the condensing electrode and theremaining waste-product has been exchanged with the reference solution,the frequency of the applied electric field is then changed (generallyfrom high to low) so that the dielectrophoretic force changes sign andbecomes repulsive, and the analyte is then released into a small (˜1 μl)volume of the reference solution. Next, the analyte is transported tothe sensor chamber and restricted in the vicinity of the sensor.Detection is then performed by sensor arrays, selectively functionalizedfor the target analyte (bacteria) of interest.

Microfluidic separator 16 is comprised of a high throughput system ofmultiple microchannels, preferably an array 10×100 microchannelsalthough any number of microchannels may be utilized with varyingdegrees of efficiency. FIG. 2 is a schematic view of a microchannelassembly 30 having a plurality of individual microchannels 32. Eachmicrochannel assembly 30 has multiple-outlet linear microchannels 32with copper/metal sets of electrodes 34 deposited on the microchannelwalls. In a preferred embodiment, microfluidic microchannel assembly 30includes plates 36 patterned with metal electrodes 34, such as copperand the like, on each side, generally having a preferred geometry of 5μm×10 mm×1 μm, sandwiching an internal channel structure or “tree” 38outlined with copper electrodes 34. When plate 36 comes in contact withinternal channel structure 38, multiple microfluidic channels areformed. Plates 36 and internal channel structure 38 may comprise plasticmaterial or other light, durable material capable of securing metalelectrodes, and containing the test sample without degradation.

An additional electrode design is introduced for high yield pathogen(bacteria and cell) capture, and which lends itself to enhanced andreliable separation using an alternating electric field.

The capture of pathogens is generally from blood, water, and otherfluids, and the separation includes the removal of pathogens from, forexample, large blood components, red blood cells, white blood cells, andthe like.

The purpose of optimizing the electrode design for this application isto maximize the yield for a given applied voltage. FIG. 18 depicts anembodiment of the electrode design 200 for use within the confines of amicrochannel. Electrode 200 is composed of a set of densely packedconductive rings fragments 204, 206. The rings may be connectedseparately to a voltage source or multiple voltage sources, orinterconnected and attached to a single voltage source. Electrode 200may include concentric rings, connected rings, or a spiralconfiguration. An embodiment depicting a set of adjacent concentricrings is shown in FIG. 19. In this embodiment, the rings are coaxial,extending radially outwards from a center point 202. The innermost orfirst ring 204 a may be electrically connected to the third ring 204 b,which may be electrically connected to the fifth ring 204 c, and so onfor the odd numbered rings extending radially from center point 202.Each of the odd numbered rings are either electrically connectedtogether to have a same first voltage and polarity, for example V+, orconnected separately to at least one voltage source having the samevoltage and polarity. In a similar fashion, the even numbered ringsextend radially from center point 202 and may be electrically connectedtogether. As depicted in FIG. 18, second ring 206 a may be electricallyconnected to the fourth ring 206 b, which are connected to a singlevoltage source having a second polarity, for example V−, the secondvoltage being different from the first voltage in either magnitude,polarity, or both. Each of the even numbered rings are eitherelectrically connected together to have a same second voltage andpolarity, or connected separately to at least one voltage source havinga second voltage and polarity.

An alternating current (AC) electric field is applied to the electrodeat a predetermined frequency or frequencies in the range of 1 kHz to 400MHz, such that the resulting force acting on the different speciesallows for a differential response. The method can be applied forseparation of similar size particles also from fluids other than blood.

FIG. 20 depicts the alternating polarities associated with a positivevoltage source connected to one set of electrode rings, and a negativevoltage source connected to another set of electrode rings, where eachring of each set is concentric and alternates radially outwards from thecenter point, one set representing the odd numbered rings and the otherset representing the even numbered rings.

FIG. 21 depicts a typical pathogen contaminated fluid inside amicrochannel of the present invention. Red blood cells 210 and E. coli212 are present in the fluid. FIG. 22 depicts the same microchannelcontaining contaminated fluid with the electric field ON. As can beseen, the E. coli 212 aligns with the curvature of the electrodes underthe dielectrophoresis force. This force “holds” or redirects thepathogen while the carrier fluid (blood) traverses through themicrochannel.

As depicted by FIG. 23, a positive dielectrophoresis force (pDEP) of 3volts at 10 MHz was shown to align the pathogens effectively andefficiently. This is in stark contrast to a negative dielectrophoresisforce (nDEP) of 3 volts at 200 MHz as depicted in FIG. 24. Thisillustrates the frequency dependence of establishing pathogen attractionor repulsions.

Preferably, voltages of opposite polarity are applied to adjacent rings,or a different voltage level is applied to adjacent rings, such thatthere exists a potential difference between adjacent rings. Thus, eitherV₁=−V₂, or there is a phase difference between the two voltage sources(V1=Vsin (ωt); V2=Vsin (ωt+π)), or the magnitude of V₁ is not equal tothe magnitude of V₂.

FIG. 3 is a schematic view of an array 40 of microfluidic microchannelassemblies 30 of the present invention used for separation. Theinvention provides for an assembly of layers with defined components ofmultiple microchannels. Microchannel assemblies 30 are stacked such thatarray 40 comprises an “m×n” microchannel array. Each microchannel 32 hasa height h and width w. In a preferred embodiment, each microchannel 32is approximately 100 μm wide and 100 μm high. The preferred length ofeach microchannel 32 is 10 mm. Other dimensions may be pre-determinedfor particular efficiencies and for specific test samples. The advantageof an array of microfluidic channels is the ability to transport andseparate the test sample in an extremely small package—on the order ofan integrated circuit. The chip-set size of the filtration systempromotes reliability, portability, and discrete packaging.

In a preferred embodiment, array 40 is composed of multiple plates 36sandwiching internal channel structures 38 such that when stacked theyform an array of 10 (horizontal)×100 (vertical) microfluidic channels.The 11 layers are aligned to the edges and thermally bonded.

In an assembled microfluidic separator 16, laminar flow conditions areprovided for separation. Under preferred operating conditions, flowvelocity, v, is approximately 100 μm/s, channel length, L, is about 1cm, the total flow time through a single channel, t_(flow), is onaverage about 100 s, and the flow rate per channel is about 1 nL/s.Thus, in total 1 cc is pumped very quickly through 1000 microchannels.

The test sample throughput may be “tuned” by increasing or decreasingthe number of parallel microchannels, increasing or decreasing theparallel stacked microchannel assemblies 30, and changing the flowvelocity by setting the pumping speed.

The invention utilizes the frequency dependence of the sign of the CMfactor between different contaminant/blood species for separation in aseries of custom designed dielectrophoretic modules.

For the purposes of the present invention, a coefficient α will bedefined as follows:

α=2πε_(m) r ³ Re{CM(ω)}; and

F_(DEP)=α·∇E²;

where CM(ω) is as defined previously.

The α coefficient accounts for the particle size r and the dielectricproperties of the particle itself ε_(p) and the surrounding mediumε_(m).

Thus, the invention provides for separation of species based on thedifferent signs of the Re{CM(ω)} factor and which follows the differentsigns of the α coefficient at a chosen operating frequency. Theseparation method of two select groups of the components of interest,i.e., pathogens/cells/bacteria/particles (group 1) and blood cells(group 2), is based on tuning the electric field frequency such that theRe{CM(ω)} factor is positive for one group and negative for the othergroup. This causes component movements in different directions, whichleads to separation. Unlike the prior art, the microfluidic separator isuniquely designed to complete separation, and the subsequent condensingand flushing process steps result in pure isolation of the bacteria,cells, or particles of interest.

A description for bacteria in blood is provided below for theseparation, capture, and release mechanisms, and for flow conditionsinside a microfluidic channel. Figures of force fields are generatedfrom program runs using COMSOL Multiphysics software.

An electric field gradient is generated by an electrical waveformapplied to sets of electrodes on the plurality of microchannel walls.FIG. 4 depicts a cross-section of microchannel 32 with ground electrodes42 and waste collecting electrode 44. In this example, 20 volts areapplied to waste collecting electrode 44. The resultingdielectrophoretic forces F_(DEP) acting on bacteria are depicted byarrows 46 in FIG. 5, while the dielectrophoretic force acting on redblood cells are shown by arrows 48 in FIG. 6. These forces indicate thatF_(DEP) for bacteria has an opposite direction from F_(DEP) for redblood cells.

FIG. 7 is a cross-sectional top view of a microchannel 32 showing thetrajectories of the test sample components. Trajectories of bacteria 50and red blood cells 52 in the direction of flow 54 are depicted forpre-determined electrode geometry inside one of the many microfluidicchannels 32 that comprise microfluidic separator 16. Under the appliedwave form, bacteria and red blood cells are pushed towards opposite endsof the microchannels.

As an illustrative example, the values of α are selected for E. colibacteria, red blood cells, and white blood cells based on pre-determinedpermittivity data (real and imaginary permittivity ε_(p), ε_(m),particle radius r, and conductivity σ).

The dielectrophoretic force acting on E. coli bacteria, red blood cells(RBC), and white blood cells (WBC) is generated by applying a voltage,which in the preferred embodiment is approximately 20V, on theelectrodes at different operating frequencies. The provided values ofthe coefficient α for RBC, WBC, and bacteria at 10 MHz in blood serumare shown in Table I identified in FIG. 8.

In Table I (FIG. 8), the real and imaginary part of the complexdielectric function, conductivity, coefficient α, and the real part ofthe Clausius-Mossotti factor are calculated at 10 MHz for two differenttypes of bacteria (E. coli and Micrococcus), white blood cells (Tlymphocytes, monocytes, B lymphocytes, and granulocytes), and red bloodcells.

In this example, α is negative for bacteria and positive for the bloodcomponents, thus effecting separation under dielectrophoretic force. Atan electric field frequency of 10 MHz, and using blood serum as asurrounding medium, bacteria (E. coli and Micrococcus), experience anegative dielectrophoretic force, while at the same operating conditionsthe blood components, WBC and RBC experience a positivedielectrophoretic force. Bacteria and red blood cell trajectories uponapplied dielectrophoretic force in the microchannel are depicted in FIG.9. Arrows denote the direction of the dielectrophoretic force acting onred blood cells and bacteria. The time based trajectory of motion fordifferent initial positions of red blood cells is depicted in boxes a,b, and c. The time based trajectory of motion for different initialpositions of bacteria is depicted in boxes d, e, and f. As shown, redblood cells initially positioned near microchannel walls far from theblood cell collecting electrode reach the blood cell collectingelectrode in less than one hundred seconds. In the same electric field,bacteria are pushed away from the blood cell collecting electrode anddirected towards the channel medium.

The dielectrophoretic force acting on red blood cells is directedtowards the field maximum, where the waste collecting electrode isplaced. The dielectrophoretic force confines bacteria within a certain“safe” region of the microchannel as shown in FIG. 9, boxes d, e, f,while it pushes blood cells in the opposite direction, which is towardsthe waste collecting electrode and the waste channels as shown in FIG.9, boxes a, b, c. In this manner, separation occurs continuously duringtest sample transport through the microfluidic channels, with eachmicrochannel doing its part to separate test sample components.

In the current example, utilizing the preferred array geometry for themicrofluidic separator array with lateral and vertical DEP electrodes,the provided separation efficiency of E. coli from RBC and WBCcomponents was nearly 95% in about 15 seconds, and 100% for anapproximately 100 micron channel length in a timeframe of approximatelyone minute. The microfluidic separator comprising an array ofmicrofluidic channels, each acting to separate the test sample anddirect waste- product towards a waste chamber.

Unique to the present invention, a branched microfluidic design allowsfor separated components to be discarded as waste, while the target ofinterest, for example E. coli, is transferred to a condenser, flushed,and then localized for pathogen detection by an electronic sensor. Theinvention is not dependent upon a single critical dimension fabricationor alignment, and the waveform frequencies may be tuned to change thedifferential sign of the Re{CM(ω)} factor for different components to beseparated. The cross-over frequency varies for different particles,bacteria, and/or cells in different media.

The values of the α coefficient for bacteria E. coli and Micrococcus inbuffer solution and blood serum at frequencies 10 MHz and 400 Mhz areprovided in Table I of FIG. 8 and Table II of FIG. 10. In Table II (FIG.10), the pre-determined values for the real and imaginary permeabilityas well as the particle radius and conductivity are listed. To enhanceseparation efficiency a pre-determined waveform containing frequencycomponents tuned for particular species (particles/bacteria/cells) ofinterest is used.

Continuing with the example above, the α coefficient for E. coli andMicrococcus is negative and has a different magnitude in blood serum at10 MHz, which for E. coli α=−0.0044(10⁻²⁴) J(m/V)², and for Micrococcusα=−0.0027(10 ⁻²⁴) J(m/V)², while the α coefficient is positive and has asimilar magnitude in blood serum at 400 MHz (E. coli α=0.0044(10⁻²⁴)J(m/V)², Micrococcus α=0.0043(10⁻²⁴) J(m/V)²).)) Micrococcus and E. coliwill experience a very similar force in blood serum at 400 MHz, whilethey will experience a very different (opposite) force in the samemedium, blood serum, at a frequency of 10 MHz.

The α coefficient for T. Lymphocytes is positive (α=0.0136(10⁻²⁴)J(m/V)²) in blood serum at 10 MHz. Thus, the DEP force (negative DEP)exerted on bacteria in blood serum at 10 MHz has an opposite sign thenthe DEP force (positive DEP) exerted on T. Lymphocytes in blood serum at10 MHz.

Consequently, a waveform applied to the electronic device of the presentinvention, containing only a frequency component at 400 MHz will resultin a very similar behavior of both E. coli and Micrococcus, causingsimilar motion of both products. A waveform applied to the electronicdevice containing only a frequency component at 10 MHz will result in asimilar motion of both E. coli and Micrococcus, and this motion will bein the opposite direction of T. Lymphocytes.

A waveform applied to the device containing both frequency components 10MHz and 400 MHz will result in a motion of Micrococcus while the forcewill cancel for E. coli, resulting in a lack of motion of E. coli.

A choice of a waveform in the same medium allows differentiating andfingerprinting different species. Unique to the present invention, asequence of an array of modules with tuned waveforms would allowselecting species based on their unique dielectric function.

After passing through the segments of microfluidic separator 16, thefirst component of the filtration system, the targets of interest (e.g.,bacteria) are separated from pollutants (e.g., blood cells), at whichpoint, the targets of interest are then condensed by condenser 20.

In a preferred embodiment, condenser 20 uses the change of the Re{CM(ω)}factor upon the change of the medium permittivity (ε_(m)) for speciescapture on a capturing electrode, to reduce the volume of the sample andcondense the species bacteria, cells, and/or particles in asignificantly lower volume. A collecting electrode attracts the bacteriato the inlet of a microfluidic sensor 26 containing sensor arrays.Movement of bacteria to a field-effect-transistor based sensor isenhanced using the electric field and the dielectrophoretic force toovercome the diffusion limitation of the motion.

FIG. 11A depicts a graph of a simulation of the dielectrophoretic forceon E. coli inside a microfluidic channel as a function of channelposition and travel time. FIG. 11B depicts a graph of a simulation ofthe dielectrophoretic force on RBCs inside a microfluidic channel as afunction of channel position and travel time. These simulation resultsshow that bacteria, if placed within 10 μm of the elimination electrode,are repelled towards the safe zone within 0.007 seconds. For RBCs, theelimination time is shorter than 82 seconds. Fifty percent (50%) of theRBCs are filtered out within the first 0.5 seconds. Ninety-five percent(95%) of the RBCs are filtered out within the first 16 seconds, andsubstantially all of the RBCs are filtered out within the first 82seconds.

At a matching frequency, the Re{CM(ω)} in the medium surrounding thespecies is positive, which results in a positive dielectrophoretic forcedirected towards a capturing electrode. The set of electrodes and theirgeometry in the microchannel cross-section is shown in FIG. 12 forcapturing and immobilizing bacteria. The direction of thedielectrophoretic force acting on bacteria is shown in the force diagramof FIG. 13. Arrows 60 show the direction of the dielectrophoretic forceon bacteria, causing the bacteria to collect on the electrodes. Despitethe flow directed towards the microchannel outlet 63, bacteria arecollected on the collecting electrodes 62 due to a positivedielectrophoretic force and a positive Re{CM(ω)} factor.

FIG. 14 depicts a flow velocity profile 64 with inflow from left and thedielectrophoresis force directing/pushing bacteria to the sensor at thebottom of a microfluidic channel (overcoming diffusion limitations).Arrows show direction of the dielectrophoretic force acting on bacteria68. Despite the flow directed towards the microchannel outlet, bacteriaare collected on the collecting electrode due to a positivedielectrophoretic force and a positive Re{CM(ω)} factor. When thesurrounding medium is changed by the buffer solution, the value of theRe{CM(ω)} factor becomes negative, and the dielectrophoretic forcerepels bacteria from the electrode causing species release.

To enhance separation, overcome the limitation caused by high ionicstrength of the solution, and obtain functional analyte (bacteria)response, the initial medium (e.g., blood serum) is diluted andpartially replaced by the buffer solution. As a result, the dielectricconstant of the medium ε_(m) changes and the Re{CM(ω)} factor changesresulting in a change of the magnitude and potentially direction of theforce.

The change of the Re{CM(ω)} factor upon the change of theparticle/cell/bacteria permittivity (ε_(p)) is used to obtain adifferential functional response.

This form of α-screen testing allows for a portable platform for rapidmultiplexed analyte detection, such as bacteria, from blood samples ofill patients at a point-of-care application. Doctors would be able todiagnose the bacteria of infection, and accurately prescribe only thenecessary antibiotic, resulting in a more efficient disease treatment,and limiting antibiotic-resistance formation.

Using the apparatus of the present invention, this a-screen testing doesnot require additional laboratory space, and is low in energyconsumption. It may be used with a sensor network integrated with foodprocessing lines in food processing plants for continuous food productquality monitoring, or used in food storage and transport. It may beintegrated in a hand-held unit for rapid Vibrio cholera and E. colibacteria detection from water samples to determine water safety.

By introducing to the medium a reference solution, such as a buffer, andadditional pharmaceuticals, the dielectric constant of the medium ε_(m)changes, the dielectric constants of the particles/bacteria/cells ε_(p)change, and Re{CM(ω)} change for different species, resulting in achange in F_(DEP) allowing to distinguish between the analytecomponents.

Using the previous values as an illustrative example, the α coefficientfor E. coli and Micrococcus is negative and has a different magnitude inblood serum at 10 MHz (E.coli α=−0.0044 (10⁻²⁴) J(m/V)² , Micrococcusα=−0.0027 (10⁻²⁴) J(m/V)²), while α is positive in a PBS buffer solutionat 10 MHz (E.coli α=0.0055 (10⁻²⁴) J(m/V)² , Micrococcus α=0.0106(10⁻²⁴) J(m/V)²}. The force F_(DEP) on Micrococcus in serum will have alower magnitude than on E. coli; however, in a buffer solution (such asPBS) the force on Micrococcus will be stronger than on E. coli.Introducing a pharmaceutical or a substance (antibiotic) which piercesonly the membrane of alive Micrococcus at 10 MHz in PBS, but not themembrane of E. coli or dead Micrococcus in buffer will allowdifferentiating alive from dead Micrococcus and E. coli, since thedielectrophoretic force depends on the size of theparticle/bacteria/cell, where F_(DEP) is proportional to r³.

Thus, a tuned chemical modification of the medium allows differentiatingand fingerprinting different species. A sequence of modules with tunedchemical modifications will allow species selection.

In the preferred embodiment, the invention applies an electricalwaveform and a dielectrophoretic force for enclosing the separatedbacteria in a small volume around a sensor to significantly decreasediffusion time to the sensor. Bacteria trapping on a nanowire ornanoribbon sensor is a resultant of the dielectrophoretic trappingmechanism and surface modification of the sensor for capture. In thismanner, a dielectrophoretic force is used as a confining force fortrapping micro-sized blood components (RBC, WBC, bacteria, and thelike).

The DEP capture mechanism for bacteria decreases the volume of diffusionof a product of interest (particle, bacteria, and/or cell) in the sensorchamber, and decreases the time for the product of interest to diffusetowards the sensor surface, which is necessary for detection.

The electronic device that implements this separation may beminiaturized to an integrated circuit, and does not require trainedpersonnel—the user only introduces a sample (such as blood or water)into the inlet chamber, and an automated process performs sampling,separation, condensation, transport, and detection. Usingdielectrophoresis, the device automatically separates any presentbacteria from the rest of the sample—for example, with blood, the largeblood components (e.g., red and white blood cells). The separatedbacteria are concentrated by a second dielectrophoretic region, andfinally detected using label-free nanosensors which may befunctionalized with bacteria specific antibodies for selectivity.

The levels of integration of the present invention on an integratedcircuit chip are generally depicted by the fabrication steps of FIG.15A. Fig. Step A depicts a printed circuit board with an integratedcircuit 69, embedded electrode connections 70, 71 and an embedded sensor72. The subsequent layers and components depicted in Steps B-G arestacked consecutively and thermally and/or chemically bonded to form thedevice.

Step B of FIG. 15A provides a structure provided in an insulator layer73 bonded on top (or bottom) of the PCB. The opening 74 is for thesensor chamber, providing access to the sensor and embedding electricconnections. The opening 75 is for alignment of the microfluidicseparator.

Step C adds insulator layer 76 with openings for the separator, thecondenser chamber, and a microfluidic channel connecting chambers, e.g.the condenser with the sensor chamber 77.

Step D depicts the addition of the microfluidic separator module 78.

Step E adds an insulator layer forming the walls of the condenser 79, anelectrode 80, and outlet 81 from the sensor chamber.

Step F adds insulator layer 82 forming the walls of the test samplechamber 83, buffer/reference liquid chamber 84, waste chamber 85, andthe insulator layer 86 forming the walls of the separator.

Step G adds lid 87 with inlets to the chambers for sample, liquidstoring, inlets 88 to the separator, and outlet 89 from the sensorchamber and waste chamber.

FIG. 15B depicts an expanded assembly drawing of the layers representingthe fabrication steps of FIG. 15A.

FIG. 16 is an expanded assembly drawing of the layers of a microfluidicseparator. The layers include a first layer 90 having microchannelstructures and coated with a planar electrode, followed by a secondlayer 91 having discrete waste electrodes. These layers are stacked inpairs to form a microfluidic separator module 92. The interconnectedwaste collecting microchannels 93, 94 are located inside of theinsulating layers. The described assembly provides customizing thenumber of microchannels on each layer, the number of stacked layers, andthe device throughput.

In one embodiment the electrical connections and microfluidicconnections between components are provided in embedded layers as shownin FIG. 17.

FIG. 17 shows a printed circuit board 95 with embedded copperconnections 96. An integrated circuit sensor 97 is connected bywire-bonding, BGA, or flip-chip technology to the PCB. A microfluidicchannel or chamber 98 is embedded in layers of insulator 99 withopenings cut to fit the microfluidic structures 98. The layers ofinsulator 99 are stacked and thermally or chemically bonded. Theopenings and holes 100 in the layers of the insulator 99 alignvertically and form microchannels for fluid transport. The inlets andoutlets 101 to the integrated electronic microfluidic circuit aredefined in the top insulator layer 102.

While the present invention has been particularly described, inconjunction with a specific preferred embodiment, it is evident thatmany alternatives, modifications and variations will be apparent tothose skilled in the art in light of the foregoing description. It istherefore contemplated that the appended claims will embrace any suchalternatives, modifications and variations as falling within the truescope and spirit of the present invention.

Thus, having described the invention, what is claimed is:
 1. Anapparatus for pathogen detection comprising: a first chamber for storinga test sample including product to be analyzed and microscaledcomponents to be separated from said product to be analyzed; a secondchamber for storing a reference solution; a pump for pumping said testsample and said reference solution; a microfluidic separator separatingsaid product to be analyzed from said microscaled components, saidmicrofluidic separator including a plurality of microfluidic channels,each microfluidic channel including: at least one electrode forproducing a dielectrophoretic force on said test sample when said testsample is pumped through said microfluidic channel to perform adielectrophoresis-based separation, said at least one electrodecomprising: a plurality of concentric rings or concentric arcs extendingradially outwards from a center point, electrically connected to avoltage source such that when voltage is applied to said at least oneelectrode, said concentric rings or concentric arcs alternate in voltagepotential, wherein each odd numbered ring or arc counted from saidcenter point is held to a first voltage potential, and each evennumbered ring or arc is held to a second voltage potential, said firstvoltage potential different from said second voltage potential inmagnitude, phase, polarity, or any combination thereof; and channels fortransporting said microscaled components away from said product to beanalyzed.
 2. The apparatus of claim 1 including: a third chamber forstoring said microscaled components when separated from said product tobe analyzed by said plurality of microfluidic channels; a condenser forcapturing said product to be analyzed once said product has passedthrough said microfluidic channels and is substantially separated fromsaid microscaled components; and a sensor for detecting said product tobe analyzed.
 3. The apparatus of claim 1 wherein said plurality ofmicrofluidic channels are assembled in an array, each microfluidicchannel having said at least one electrode on an internal wall fordelivering said dielectrophoretic force to said test sample traversingthrough said microfluidic channel, or delivering forces arising from ACelectric fields to said test sample, or a combination thereof.
 4. Theapparatus of claim 3 wherein said microfluidic channels comprise aplurality of plates, such that each microfluidic channel represents anelongated pathway for said test sample capable of providing adielectrophoretic force to said test sample as said test sampletraverses said microfluidic channel.
 5. The apparatus of claim 4 whereinsaid plurality of plates and said plurality of internal channelstructures are combined to form said array of microfluidic channels. 6.The apparatus of claim 2 including a collecting electrode to attractsaid product to be analyzed at an inlet of said sensor.
 7. The apparatusof claim 2 wherein said sensor includes a nanowire sensor, nanoribbonsensor, or ion sensitive field effect transistor, and is capable ofapplying a confining dielectrophoretic force, trapping said product tobe analyzed.
 8. The apparatus of claim 1 wherein said pump comprises amicro-pump operating in tandem with micro-valves to achieve a fullyautomated pathogen detection filtration system capable ofminiaturization to a chip-scale design.
 9. The apparatus of claim 1including a microfluidic transport module for transporting said productto be analyzed to a location in the vicinity of a sensor.
 10. Theapparatus of claim 1 wherein said even numbered concentric rings or arcsare in electrical communication with one another, and said odd numberedconcentric rings or arcs are in electrical communication with oneanother.
 11. An apparatus for pathogen detection comprising: amicrofluidic assembly including a plurality of microfluidic channelsforming an array, at least one of said microfluidic channels including:at least one electrode for establishing electrophoretic forces on a testsample separating portions of said test sample into an analyte and awaste product, said at least one electrode comprising a plurality ofconcentric rings or concentric arcs extending radially outwards from acenter point, electrically connected to a voltage source such that whenvoltage is applied to said at least one electrode, said concentric ringsor concentric arcs alternate in voltage potential, wherein each oddnumbered ring or arc counted from said center point is held to a firstvoltage potential, and each even numbered ring or arc is held to asecond voltage potential, said first voltage potential different fromsaid second voltage potential in magnitude, phase, polarity, or anycombination thereof; and adjacent microchannels for receiving said wasteproduct attracted by a dielectrophoretic force, removing said wasteproduct from said analyte.
 12. The apparatus of claim 11 wherein atleast one of said microfluidic channels comprises a plurality of plates,and a plurality of internal channel structures having at least oneinternal channel structure patterned with said at least one electrodeconfiguration, such that each microfluidic channel represents a pathwayfor said test sample capable of providing a dielectrophoretic force tosaid test sample as said test sample traverses said microfluidicchannel.
 13. The apparatus of claim 12 wherein said plurality of platesand said plurality of internal channel structures are combined to formsaid array of microfluidic channels.
 14. The apparatus of claim 11wherein said even numbered concentric rings or arcs are in electricalcommunication with one another, and said odd numbered concentric ringsor arcs are in electrical communication with one another.
 15. Theapparatus of claim 11 including a condenser having an electrode forlocalizing said analyte for sensing; and a sensor for detecting saidanalyte.